Epstein-Barr virus nuclear antigen 3B- and 3C-regulated cellular genes in virus-immortalized B cells

The cellular genes and pathways that Epstein-Barr virus (EBV) manipulates to facilitate lifelong persistence in infected hosts and efficient transmission to new hosts are not well understood. The EBV Nuclear Antigen 3 (EBNA-3) family of latent infection proteins are transcriptional regulators that influence viral and cellular gene expression in EBV-infected cells. One member of this family, EBNA-3B, is believed to be dispensable for virus replication and virus-mediated B cell immortalization in vitro , and thus is likely to regulate cellular genes that provide biological benefits for virus infection in vivo . The hypothesis that EBNA-3B is dispensable in vitro was based on 3 EBV mutants, 2 of which were known to retain significant EBNA-3B coding sequence. The EBNA-3B gene in the third mutant was sequenced and revealed to contain an in-frame 263 as deletion, showing that none of the known EBNA-3B mutants were completely deleted for EBNA-3B. Subsequent deletion of the entire EBNA-3B gene from a molecular EBV bacterial artificial chromosome clone (3B - ) had no adverse impact on the growth of virus-immortalized lymphoblastoid cell lines (LCLs). Another EBNA-3B deletion mutant was constructed where the resultant LCLs grew more slowly and exhibited decreased EBNA-3C expression in addition to a lack of EBNA-3B expression (3B - /3C low ). Transcriptional profiling of these LCLs and confirmation of the results by quantitative RT-PCR in both 3B - /3C low and 3B - LCLs revealed 5 EBNA-3C-regulated, and 3 EBNA-3B-regulated cellular genes. Jagged1 was an EBNA-3C-repressed cellular gene, and increased Jagged1 expression on 3B - /3C low LCLs could induce functional Notch signalling. Inhibition of Jagged1-induced Notch signalling had no effect on LCL growth, suggesting that EBNA-3C-mediated Jagged1 repression does not play a significant role in regulating EBV-induced B cell proliferation. CXCR4 was an EBNA-3B-repressed cellular gene, and increased levels of CXCR4 expression on 3B - LCLs resulted in increased migration in response to ligand. Stable EBNA-3B expression in 3B - LCLs could repress CXCR4, and this activity was dependent on RBP-Jκ/CSL binding. Thus two EBV-regulated cellular genes have been identified which are not involved in EBV-induced B cell growth, opening the door for further studies on ways in which they could contribute to successful virus infection in vivo .